A mutant hunt for defects in membrane protein assembly yields mutations affecting the bacterial signal recognition particle and Sec machinery - PubMed (original) (raw)

A mutant hunt for defects in membrane protein assembly yields mutations affecting the bacterial signal recognition particle and Sec machinery

H Tian et al. Proc Natl Acad Sci U S A. 2000.

Abstract

We describe an Escherichia coli genetic screen that yields mutations affecting two different cellular processes: disulfide bond formation and membrane protein assembly. The mutants defective in disulfide bond formation include additional classes of dsbA and dsbB mutations. The membrane protein assembly defective mutants contain a mutation in the secA operon and three mutations in the ffs gene, which encodes 4.5S RNA. These latter mutations are the only ones to be isolated in a gene encoding a component of the bacterial signal recognition particle by screening in vivo for defects in membrane protein insertion. A sensitive method for examining membrane protein localization shows that the ffs and secA locus mutations affect membrane assembly of the polytopic membrane protein, MalF. The ffs mutations also affect the membrane insertion of the FtsQ and the AcrB proteins. Although both the ffs and the secA locus mutations interfere with membrane protein assembly, only the latter also reduces export of a protein containing a cleavable signal sequence.

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Figures

Figure 1

Figure 1

The MalF-LacZ102 fusion protein and its topology in the wild type and the proposed localization of the fusion protein when cells are defective in the formation of disulfide bond or membrane protein insertion.

Figure 2

Figure 2

The predicted 4.5S RNA structure. The three types of mutations are listed below the wild-type sequence. The wild-type sequences corresponding to the mutation sites are underlined.

Figure 3

Figure 3

Detection of the MalF-PSBT fusion proteins in wild type and the three ffs mutants. The MalF-PSBT periplasmic fusions I and J and cytoplasmic fusions K and L were expressed from the pGJ78 plasmid series (I through L) in either the wild type (HPT183) or the three ffs mutant strains (_ffs_29 in HPT184, _ffs_69 in HPT185, and _ffs_91 in HPT186).

Figure 4

Figure 4

Protein secretion is defective in the gene X mutant, but not in the ffs mutant. Pulse–chase and immunoprecipitation of OmpA were carried out as described. The wild type (JP313), the strongest ffs mutant (_ffs_69 in HPT244), and the gene X mutant (HPT265) were examined for the efficiency of the secretion pathway.

Figure 5

Figure 5

ffs69 mutation causes defects in the insertion of FtsQ and AcrB into the membrane. (A) Detection of the FtsQ-PSBT (expressed from pHP42) in the wild type (HPT242) and the strongest ffs mutant (_ffs_69 in HPT245). (B) Detection of the AcrB576-PSBT (expressed from pHP44) in the wild type (HPT183) and the strongest ffs mutant (_ffs_69 in HPT185).

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